Project Summary/Abstract Obesity has reached epidemic proportions and is tied to the greater prevalence of metabolic disorders such as diabetes and cardiovascular disease. While the precise mechanisms by which obesity causes diabetes are not entirely clear, mounting evidence suggests that the body?s normal process of sensing lipids is disrupted. This inability to properly detect lipids can lead to lipotoxicity and cause detrimental effects in key insulin sensitive tissues. Thus, an important scientific goal, and that of this NIH Pathway to Independence Award, is to understand the mechanisms by which cells sense lipids and thereby maintain lipid homeostasis. The training component of this application builds upon the candidate?s interest and background in imaging metabolism and metabolic signaling/energy sensing, while providing a unique environment to train in career development activities related to a team science approach of doing research. The research training component will utilize a unique set of tools that will allow the candidate to probe the direct effects of lipolysis independent of transmembrane-protein kinase A (PKA) signaling and image fatty acid metabolism. The candidate will gain experience in global analysis techniques of phosphoproteomics and lipidomics, and super-resolution imaging. Utilizing these tools and training, the candidate will determine 1) the signals directly generated by lipolysis, and 2) the dynamics of lipid trafficking and lipolysis-derived signals within a cell. Central to this aim is the hypothesis that signals directly produced by lipolysis function to maintain lipid homeostasis and are highly dynamic. The research component of the award will be accomplished by the following specific aims: Aim 1: Identification of signals that are generated directly by lipolysis. Lipolysis is known to produce signals, but up to this point the direct effects of lipolysis were not distinguishable from transmembrane-PKA signals. Utilizing novel synthetic ligands that activate ABHD5, a lipase co-activator protein, Aim 1 will be accomplished by the following sub-aims: 1a: To identify ABHD5-dependent lipid mediators. Utilizing a lipidomic approach, the candidate will identify the bioactive lipids produced by ABHD5 that regulate downstream metabolism. 1b: To identify ABHD5-dependent kinase activation pathways. Using a phosphoproteomic approach, the candidate will determine the phosphorylation events, kinases and pathways that are a direct consequence of ABHD5 activation. Aim 2: To determine the trafficking dynamics of fatty acids and their metabolites and lipid mediators. This will be accomplished by the use of newly developed genetically encoded fluorescent sensors that allow the monitoring of the temporal and spatial dynamics of fatty acids and fatty acyl-CoAs. The proposed K00/K99 is well aligned with the mission of the NIH and the NIDDK and will train a promising scientist to understand the mechanisms that regulate lipid homeostasis and potentially the pathways that are disrupted during obesity, a significant public health priority.